The long term objective of this proposal is to provide an understanding of the genetic and biochemical mechanisms involved in cellular resistance to ionizing radiation. The general approach taken will be to use gamma-ray sensitive Chinese hamster mutants to isolate human genes which complement the radiation sensitivity of these mutants. Currently, we have isolated 12 mutants which have increased sensitivity to killing by gamma-rays. In this proposal, we will focus on isolating an characterizing a human repair gene located on chromosome 5 which complements the double-strand DNA break repair defect and gamma-ray sensitivity of one of these mutants, XR-1. Two gamma-ray resistant XR-1: human hybrid cell lines have been obtained that have a limited amount 2 Mb of human DNA. DNA probes from this hybrid will be generated using the polymerase chain reaction (PCR) with primers directed to human repetitive sequences. Probes which are common to both hybrids will be used to screen a yeast artificial chromosome (YAC) human DNA library and obtain a set of YACs which contain the human DNA inserted into the gamma-ray resistant hybrid. These YACs will be introduced into the XR-1 cell by spheroplast fusion and tested for their ability to confer gamma-ray resistance. Using these YACs as probes, a set of cDNAs coding for the inserted human DNA will be obtained by screening cDNA libraries. Full length cDNAs will be constructed and candidate cDNAs containing the resistance gene identified by transfection into XR-1 and selection for gamma-ray resistance. The DNA sequence of the cDNA clone will be obtained and the extent of sequence homology with other known repair genes determined.